1. Field of the Invention
This invention describes a method for catalyzing oxidation reactions, reduction reactions, or both using free radical intermediates. More specifically, this invention involves the use of biologically derived peroxidases in the generation of a variety of oxidation or reduction agents consisting of cation radicals, anion radicals, neutral radicals, or oxygen radicals. Such oxidation and reduction agents can be employed separately or in combination to carry out a wide variety of oxidation or reduction reactions, some of which involve the degradation of recalcitrant organic compounds such as organic environmental pollutants.
2. The Relevant Technology
The use of oxidation or .reducing agents to carry out oxidations or reductions on targeted substrates is mature technology well-known in the art. Desired oxidation and reduction reactions can be carried out on a multitude of different substrates simply by reacting the substrate with a stoichiometrically adequate amount of an appropriate oxidant or reductant. Commonly used oxidants or reductants which can be produced in a commercially feasible manner include a wide variety of generally inorganic agents. The feasibility of using such oxidants or reductants is often limited by such restraints as the cost of the reactant in relation to the value of the reacted substrate, the ability to control the reaction, and the ability to obtain suitable concentrations of the reacted substrate in reasonably pure amounts.
More complicated oxidation and/or reduction reactions have been created which involve organic intermediates, such as hydroquinones, alkylanthraquinones, anilines, hydrazines, or metal complexed chelating agents. In some cases, the reactant is a catalyst which is continuously regenerated. For example, U.S. Pat. No. 5,143,710 to Sawyer et al. discloses methods for generating superoxide ions in situ catalyzed by aniline, N-substituted aniline compounds, or phenylhydrazine compounds. The superoxide ion, which is an anion radical, is useful for a number of different applications discussed within Sawyer et al. Superoxide ions have proven particularly effective in destroying a variety of halogenated hydrocarbons such as polychlorinated biphenyls ("PCBs") and similar toxic materials. In general, superoxide ions are useful reducing agents.
U.S. Pat. No. 3,998,936 to Ernst et al. discloses a process for regenerating the activity of the catalyst used in the hydrogenation (or reduction) stage of the cyclic anthraquinone process for producing hydrogen peroxide involving the use of a platinum group metal catalyst. However, Ernst et al. does not disclose how an overall oxidation/reduction system could be constructed that would have broad application.
U.S. Pat. No. 4,751,068 to Bicker et al. discloses a method of catalyzing oxidation/reduction reactions of simple molecules through the redox catalytic activity of chelating agents complexed with a metal atom (the complex being referred to as a "chelate"). These chelates have been shown to be useful in converting CO and H.sub.2 O to CO.sub.2, CO and H.sub.2 S to COS, CS and H.sub.2 S to CS.sub.2, CO and NH.sub.3 to CONH, and CO and RNH.sub.2 to RNCO. However, in order to regenerate the spent chelates it is necessary to react the chelates with oxidants or reductants. No self-sustaining reaction sequence is disclosed in Bicker et al.
More recently, with the advent of more refined biochemical techniques, biologically induced oxidations and reductions have been carried out using, e.g., fungi and agents which are secreted thereby. These biologically derived reactions are often superior to simply adding oxidation and/or reducing agents to a reaction mixture because of their lower cost and greater ability to more carefully control the reaction conditions, especially those reactions which involve the use of enzymes. Enzymes have the advantage being able to overcome high reaction barriers without the input and/or generation of large amount amounts of energy such as heat. In addition, as long as the biological agent is kept alive by ensuring that the system has adequate quantities of nutrients (some or all of which are supplied by the chemicals targeted for degradation) it will continue to produce adequate quantities of the oxidation or reduction agents. In this manner, the reaction is often self-sustaining so that no new reactants need to be added to complete the oxidation and/or reduction reactions.
There are numerous examples of biologically induced degradation of organic molecules. For example, lignin, which is the structural polymer found in wood and a substance which is otherwise highly resistant to many forms of biodegradation, is readily degraded in the presence of the white rot fungus Phanerochaete chrysosporium. Kirk, T. et al., Arch. Microbiol. 117:277-85 (1978). Lignin degradation is catalyzed by a group of enzymes including extracellular peroxidases secreted by P. chrysosporium under nutrient nitrogen-limiting conditions. Gold, M. et al., Arch. Biochem. Biophys., 234:353-62 (1984); Tien, Met al., Proc. Natl. Acad. Sci. USA, 81:2280-84 (1984). It is known that both lignin peroxidases ("LIP") and manganese-dependent peroxidases are produced by white rot fungi. Glenn, J. et al, Arch. Biochem. Biophys., 242:329-41 (1985). The fungi also produce enzymes that generate hydrogen peroxide. Kelley, R. et al, Arch. Microbiol., 144:248-53 (1986); Kersten, P., Biochemistry, 87:2936-40 (1990). Veratryl alcohol (3,4-dimethoxybenzyl alcohol) is a secondary metabolite of P. chrysosporium and is also believed to be involved in lignin degradation. Harvey, P. et al., FEBS Lett., 195:242-46 (1985).
In addition, the degradation of several environmental pollutants to carbon dioxide by white rot fungi has also been reported. U.S. Pat. No. 4,891,320 to Aust et al; Bumpus, J. et al., Science, 228:1434-36; Ryan, T. et al., Appl. Microbiol. Biotechnol., 31:302:07 (1989); Fernando, T. et al, Appl. Microbiol. Biotechnol., 56:1666-71 (1990); Kennedy, D. et al., Appl. Microbiol. Biotechnol., 56:2346-53 (1990).
Although these articles generally discuss the use of white rot fungi to degrade lignin, no specific information as to the mechanism of this degradation is revealed. To the extent that certain intermediate substances such as LiP or veratryl alcohol have been shown to be involved, these articles do not contain information that would teach how to utilize white rot fungi or other useful organisms in a variety of oxidation or reduction reactions to generally oxidize or reduce any organic compound.
In the last few decades, there has been growing concern about the accumulation of toxic organic pollutants in the soil and water. Many industrial operations, particularly those involving chemical processes, have resulted in the contamination of huge amounts of soil, which in turn pollutes ground water and streams. With the fairly recent passage of stricter environmental legislation mandating the cleanup of what are referred to as "remediation sites" there has arisen a great need for practical and economically viable methods of soil and water remediation.
In the case of toxic organic pollutants such as chlorinated hydrocarbons, PCBs, and other organic solvents, the primary method of removing these from the soil involves the temporary removal of the contaminated soil, which is then passed through large columns through which hot air is passed. This causes the volatile contaminants to be driven off by evaporation. However, not only is this method extremely expensive, it does not guarantee the removal of the pollutants from the environment but simply shifts them from the ground into the air. While some degradation of these pollutants may occur in the presence of sunlight, many of the less reactive compounds are simply scattered into the air where they might later precipitate back into the earth, albeit in a more diluted form.
While organisms such as white rot fungi have been shown to degrade certain toxic pollutants in the laboratory, their use as general agents to clean up such toxic pollutants has been limited due to the lack of understanding of the reaction mechanisms involved in their oxidation and reduction. In addition, because many of the most persistent pollutants exist deep beneath the earth it has not been possible to sustain living white rot fungi or other organism cultures in the highly anaerobic conditions which exist beneath the earth. Finally, without an understanding of the necessary intermediates, or "diet" of the reactions involving the degradation of certain organic materials through the use of such organisms it has heretofore been impossible to control, or even predict, the types of organic substances that might be degraded through such mechanisms.
From the foregoing it should be understood that what are needed are compositions and methods which can be generally employed to carry out any number of oxidations or reductions on any targeted organic substrate. Moreover, it will be appreciated that it would be a significant advancement of the art if such compositions and methods could be cheaply and easily carried out by using relatively inexpensive raw materials, such as those used to grow white rot fungi.
It would yet be a significant improvement over the prior art if such compositions and methods could be varied to alternatively reduce, oxidize, or both, depending on the substrates to be degraded. Specifically, it would be a major advancement in the art if both the oxidative and reductive properties could be carefully controlled so that compounds requiring both oxidation and reduction for their degradation can be fully degraded utilizing a single reactive system, or different systems or conditions in series.
It would yet be a significant improvement over the prior art to provide compositions and methods under a variety of conditions which could degrade a variety of recalcitrant environmental pollutants such as PCBs, chlorinated hydrocarbons, and other toxic organic wastes without having to physically alter the reaction conditions once the reactions are set in motion. In addition, because living organisms are typically employed to carry out these reactions, it would be a major advancement in the art if such compositions and methods resulted in the generation of sufficient molecular oxygen so that the organisms would stay alive even under extremely anaerobic conditions, such as in remediation sites where the organisms are injected deep into the contaminated soil.
Such compositions and methods are disclosed and claimed herein.